DESCRIPTION: A. actinomycetemcomitans is associated with human periodontal disease and expresses a leukotoxin (ItX) that kills human cells that are required for the immune response against infection. Most A. actinomycetemcomitans strains express low levels of this toxin, but some strains contain alterations in the ltx promoter and exhibit a hyper-leukotoxic phenotype. These strains exhibit altered regulation of the Itx genes and are associated with localized juvenile periodontitis. We will determine how the ltx genes are regulated in minimally leukotoxic organisms and how the regulatory processes are altered in hyper-leukotoxic organisms. We have identified a c/s-acting negative regulator of toxin expression that interacts with a trans-acting protein. This protein will be isolated and we will determine how this interaction reduces ltx expression. We will also determine if the acquisition of an insertion element (IS) leads to increased toxin expression by displacing this c/s-acting negative regulator upstream from the ltx genes. We will also determine if IS introduces an outwardly directed promoter that transcribes the ltx genes. We have also shown that ltx expression is induced during anaerobic growth. A c/s-acting sequence and a trans-acting polypeptide that may play a role in anaerobic regulation have been identified. We will determine if this sequence functions as an UP element that interacts with the ltx-subunit of RNA polymerase. Finally, the ltx operon possesses a fifth gene, orphan, that resides upstream from IXC. Frame shift mutations in orfA reduce ltx expression. Therefore, we will determine if the OrfA polypeptide is involved in the regulation of It expression and whether a naturally occurring deletion in orfA leads to the hyper-leukotoxic phenotype. It is clear that the leukotoxin is an important virulence determinant of A. actinomycetermcornitans and thus it is important to understand the mechanisms that lead to the hyper-leukotoxic phenotype. These studies will determine how/Ix expression is regulated in A. actinomycetemcomitans and will identify mechanisms that contribute to hyper-expression of the toxin. The cis- and/or transacting components that are found to differ in ltx hyper-expressing strains may represent new diagnostic targets to detect hyper-virulent A. Actinomycetemcomitans. It is also possible that the regulatory processes themselves may represent targets that can be exploited for the development of local or systemic therapies Aimed at affecting leukotoxin expression in vivo.